comparisons of selected household and commercial
TRANSCRIPT
University of Arkansas, FayettevilleScholarWorks@UARK
Theses and Dissertations
8-2016
Comparisons of Selected Household andCommercial Disinfectants Against PoultrySalmonella Isolates and a Survey of InternalParasites in Backyard Poultry in ArkansasKayleigh MoyleUniversity of Arkansas, Fayetteville
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Recommended CitationMoyle, Kayleigh, "Comparisons of Selected Household and Commercial Disinfectants Against Poultry Salmonella Isolates and aSurvey of Internal Parasites in Backyard Poultry in Arkansas" (2016). Theses and Dissertations. 1690.http://scholarworks.uark.edu/etd/1690
Comparisons of Selected Household and Commercial Disinfectants Against Poultry Salmonella
Isolates and a Survey of Internal Parasites in Backyard Poultry in Arkansas
A thesis submitted in partial fulfillment
of the requirements for the degree of
Master of Science in Poultry Science
by
Kayleigh Moyle
Southern Virginia University
Bachelor of Art in Biology, 2011
August 2016
University of Arkansas
This thesis is approved for recommendation to the Graduate Council.
Dr. F. Dustan Clark
Thesis Director
Dr. R. Keith Bramwell Dr. Susan. E. Watkins
Committee Member Committee Member
Abstract
Backyard and exhibition poultry have been gaining in popularity and as such there has
been a large increase in the number of small flocks. As the interaction with poultry has increased,
so has the opportunity for diseases and parasites, for both birds and people. One of the major
zoonotic illnesses is caused by the bacteria Salmonella, which can be found in commercial and
small flocks. Salmonella is the number 2 contributor of foodborne illnesses so its prevalence in
commercial flocks is of high concern. Despite improved cleaning, disinfection, and biosecurity
practices, there is still potential for disease outbreaks and infections in the industry. This thesis
concentrates on helping small flock holders fight diseases. In a two pronged approach I looked at
preventing Salmonellosis by evaluating the efficacy of 6 disinfectants, then took the opportunity
to sample and evaluate the prevalence of internal parasites in small flocks in Arkansas. Results
found that household bleach is an effective disinfectant, whereas lemon juice and vinegar are not
as effective as commercial disinfectants. Additionally, internal parasites are common in backyard
flocks in Arkansas.
Acknowledgements
I would like to thank Dr. Dustan Clark for having me as his student, and for all of the
time, effort, energy, and advice he has given me over the years. None of this would be possible
without him, and I truly appreciate everything he has done.
I would also like to thank David Cross and Patty Tyree for their help and companionship,
both inside and outside of the lab. Thanks go to Komala Arsi for always being willing to talk,
help, and teach, and to Sandip Shrestha for the late nights making plates.
Last but definitely not least, I would like to thank my parents for everything they have
done for me over the years. They have always supported and helped me, despite my
procrastination and how difficult I am to work with sometimes. I would not be where I am
without them.
Contents
Literature Review............................................................................................................................ 1
INTRODUCTION ....................................................................................................................... 2
CONTROL OF SALMONELLA ................................................................................................ 7
DISINFECTION ....................................................................................................................... 10
CLASSES OF DISINFECTANTS ............................................................................................ 14
Alcohols ................................................................................................................................. 14
Aldehydes .............................................................................................................................. 14
Oxidizing Agents ................................................................................................................... 15
Quaternary Ammonium Compounds ..................................................................................... 17
Phenols................................................................................................................................... 17
Biguanides ............................................................................................................................. 17
Halogens ................................................................................................................................ 18
INTERNAL PARASITES ........................................................................................................ 19
Ascaridia ................................................................................................................................ 19
Heterakis gallinarum .............................................................................................................. 21
Capillaria ............................................................................................................................... 21
Eimeria................................................................................................................................... 22
Comparisons of selected household and commercial disinfectants against poultry Salmonella
isolates........................................................................................................................................... 30
SUMMARY .............................................................................................................................. 31
DESCRIPTION OF PROBLEM ............................................................................................... 32
MATERIALS AND METHODS .............................................................................................. 33
Salmonella Isolates ................................................................................................................ 33
Agar Plate Preparation ........................................................................................................... 33
Disinfectants .......................................................................................................................... 34
Preparation of Bacterial Solutions ......................................................................................... 35
Standard Plate Count ............................................................................................................. 35
Efficacy of Disinfectant Against Salmonella Isolates ........................................................... 36
RESULTS AND DISCUSSION ............................................................................................... 36
Efficacy of Disinfectants Against S. Enteritidis .................................................................... 36
Efficacy of Disinfectants Against S. Heidelberg ................................................................... 38
Efficacy of Disinfectants Against S. Kentucky ..................................................................... 39
Efficacy of Disinfectants Against S. Typhimurium .............................................................. 40
Efficacy of Disinfectants Against S. Senftenberg ................................................................. 41
Efficacy of Disinfectants Against S. Montevideo ................................................................. 42
Number of Salmonella Strains Each Disinfectant Reduced/Killed at Each Time ................. 43
Discussion .............................................................................................................................. 44
CONCLUSIONS AND APPLICATIONS ................................................................................ 46
REFERENCES AND NOTES .................................................................................................. 47
A survey of the prevalence of internal parasites in chickens shown at Arkansas Fairs ................ 56
SUMMARY .............................................................................................................................. 57
DESCRIPTION OF PROBLEM ............................................................................................... 58
MATERIALS AND METHODS .............................................................................................. 58
RESULTS AND DISCUSSION ............................................................................................... 58
CONCLUSIONS AND APPLICATIONS ................................................................................ 61
REFERENCES AND NOTES .................................................................................................. 62
CONCLUSION ......................................................................................................................... 68
1
CHAPTER 1
Literature Review
2
INTRODUCTION
Commercial poultry production is the fastest growing sector of the animal agricultural
industries. Per capita consumption of chicken in the United States has risen from 36.9 pounds in
1965, to an estimated 92.1 pounds in 2016 [1]; as such, poultry production has dramatically risen
to meet demand.
Chickens that are raised specifically for meat are called broilers. As of 2015, broilers
reached an average market weight of 6.24 pounds in 48 days [2]. In 2014 there were 8.54 billion
broilers produced in the United States alone, which amounted to 51.4 billion pounds, live weight
[3]. In order to meet this demand and create supply, intensive production methods have been
developed. However, despite large improvements in health and management, there is still
potential for disease outbreaks and infections in flocks.
Keeping poultry as a hobby is one of the fastest growing hobbies in the United States.
Reasons for keeping small flocks include: egg production; meat; exhibition; insect control;
family tradition; teaching children responsibility and animal care; and companionship. Many
owners like knowing and controlling exactly what their birds eat, enabling owners to keep
certain lifestyle choices such as eating only organic food [4-5].
One of the most important bacteria to control in poultry is Salmonella, which can also
affect humans and poses a threat to public health. Salmonella is the number 2 contributor of
foodborne illnesses, second only to norovirus. Salmonella causes an estimated 11% of all
foodborne illness; additionally, an estimated 34% of foodborne illnesses resulting in
hospitalizations are caused by Salmonella. Furthermore, Salmonella has the highest number of
deaths due to foodborne illnesses, around 400 people per year [6].
Although Salmonella are a major concern in commercial poultry, they are also of concern
in small hobby flocks. Recently, backyard flocks have been blamed for 8 ongoing outbreaks in
3
the year 2016. Thus far, 611 people in 45 states have been infected; out of 493 patients
interviewed, 88% had contact with live poultry within a week before developing symptoms [7].
Salmonella are members of the Enterobacteriaceae family, and are named after USDA
veterinary bacteriologist Daniel E. Salmon. Salmonella are rod-shaped, gram-negative, non-
spore forming, predominantly motile bacteria with a diameter of 0.3-1.5 microns and a length of
two to five microns. The optimum temperature for growth is 37 C, but growth is possible in a
range of 5 to 45 C. Salmonella cannot survive above 70 C. The optimum pH is around 7, but
growth can occur anywhere between 4.0 to 9.0. They can survive in soil, litter, and water for
long periods of time, and are resistant to dehydration. Medias that supply nitrogen and carbon
can be used to culture Salmonella [8-10].
There are almost 2,500 serotypes of Salmonella identified so far, which are divided into
two species; Salmonella enterica and Salmonella bongori. S. enterica is further divided into six
subspecies: enterica (I), salamae (II), arizonae (IIIA), diarizonae (III B), houtenae (IV) and
indica (VI), which all differ biochemically. S. bongori and S. enterica subspecies II-VI are all
found in cold blooded animals and the environment. This paper will focus on S. enterica subsp.
Enterica, which is found in warm blooded animals. Out of the 2,463 Salmonella serotypes, 1,454
of them belong to S. enterica subsp. Enterica. Serotypes are differentiated based on three
antigens: the flagellar “H” antigen, the oligosaccharide “O” antigen of the outer bacterial
membrane, and the polysaccharide “Vi” virulence antigen [8, 10-11].
Salmonella serotypes can be further divided based on host specificity: host
restricted/adapted, or unrestricted. Host restricted serotypes are generally defined as being almost
exclusively associated with one particular host species. Examples of this include Salmonella
serotype Gallinarum, and Salmonella ser. Pullorum, which cause disease only in poultry. Host
4
adapted serotypes affect mainly one species but are capable of causing disease in other species,
especially those that are closely related [12]. An example of this is Salmonella ser. Choleraesuis,
which causes swine paratyphoid but is also highly pathogenic to humans [12-13]. Unrestricted
Salmonella serotypes cause disease in a wide range of unrelated hosts. Unrestricted serotypes
include Salmonella ser. Typhimurium and Salmonella ser. Enteritidis [12].
Salmonellosis in humans is acquired by consuming bacteria, usually through
contaminated foods such as poultry, eggs, beef, pork, unpasteurized milk or juice, and fresh
produce. Foods can become contaminated by contact with animal feces, by mixing raw and
uncooked meat, mixed utensils in the kitchen at home, or by cross contamination during
processing. Food may also become contaminated after coming into contact with infected animals
and not properly washing before handling food. Transmission from person to person is also
possible, through the fecal-oral route [6, 10].
Salmonellosis in humans can be divided into 3 categories: gastroenteritis (normal food
poisoning), enteric fever (typhoid and paratyphoid fever), and septicemia [10]. S. Typhimurium
and S. Enteritidis are the most common serotypes that cause gastroenteritis in the United States.
The incubation period is normally 12-72 hours, and symptoms include diarrhea, abdominal
cramps, fever, nausea, vomiting, and headache. Illness may last for 4-7 days, and most people
recover without treatment. Salmonella ser. Typhi and S. ser. Paratyphi cause typhoid and
paratyphoid fever, respectively. Salmonella Typhi and S. Paratyphi are found in and transmitted
only by humans. Septicemia occurs when Salmonella pass through the intestine and into the
blood stream. This is most often caused by invasive types of Salmonella. Young children, the
elderly, and people with compromised immune systems are more likely to have septicemic
infections than the normal populace [6, 10].
5
Diseases can be transferred two ways: from organism to organism (horizontal), or
directly from mother to embryo/fetus during pregnancy (vertical). Salmonella can be spread both
ways. Vertical transmission is especially important because Salmonella that is passed into the
eggs by layers makes its way into the human food supply, creating the potential for foodborne
illness.
There are two serovars that affect only poultry: S. Pullorum and S. Gallinarum.
Salmonella Pullorum causes Pullorum disease (PD), previously known as Bacillary White
Diarrhea (BWD). PD is found mostly in young chicks and mortality peaks during the second or
third week after hatch. Symptoms include: weakness, depressed appetite, poor growth, white
diarrhea, labored breathing, and swollen joints. Lesions in young birds usually include
unabsorbed yolk sacs and nodules in the liver, spleen, lungs, heart, gizzard, and intestine.
Pericarditis is frequently observed. If adult birds get infected with PD, there is a decrease in egg
production, egg fertility, and hatchability. Signs in adults also include anorexia, diarrhea,
depression, and dehydration [5, 8].
Salmonella Gallinarum causes fowl typhoid (FT). Fowl typhoid symptoms are similar to
those of PD, without the characteristic white diarrhea. FT also tends to infect older chicks and
adults. Sudden outbreaks may begin by a sudden drop in feed consumption; birds will be droopy,
have ruffled feathers, and the combs of the birds become pale and shrunken. Morbidity and
mortality for both PD and FT can range anywhere between 0 to 100%, depending on age, strain
of bird, nutrition, flock management, concurrent diseases, and exposure to the environment. Both
PD and FT can be transferred vertically and horizontally [5, 8].
PD was first described in 1899 and FT was recognized in 1888. These two diseases
quickly became prevalent and caused high mortality in early chicken production and flocks. In
6
1935 the National Poultry Improvement Plan (NPIP) was created in part to control PD and
eventually FT. Due to the control measures taken by the NPIP, FT and PD have been almost
completely eradicated from the United States. They are still found and cause problems in other
countries, especially those that do not have a poultry industry developed, are not developed as a
country, or are in the process of developing [8, 14].
As S. Gallinarum and S. Pullorum were eradicated, S. Enteritidis began to emerge as the
predominate Salmonella problem faced by producers, filling the niche left behind [15]. As S.
Enteritidis also causes salmonellosis in humans, it replaced S. Typhimurium as the primary cause
of salmonellosis in humans. The NPIP began to target S. Enteritidis in 1989, and along with the
use of vaccines and natural immunity development, prevalence has decreased. S.ser. Heidelberg
was the most commonly isolated serovar in poultry until 2007, when it was surpassed by S.ser.
Kentucky [15-16].
Paratyphoid infections (PT) in chickens are caused by non-host adapted Salmonella such
as S. Enteritidis (SE), S. Typhimurium and S. Heidelberg. The ability to cause disease and death
varies widely, dependent on strain, age of the bird, and amount of inoculation. PT mostly affects
newly hatched birds and can sometimes lead to illness and death at high frequencies. Typical
symptoms include: somnolence, droopiness, ruffled feathers, anorexia, diarrhea, and occasionally
blindness and lameness. Mortality reaches peak levels between 3-7 days of age. Susceptibility to
infection decreases with age. When there is infection of adult birds, most often the only symptom
is brief, mild diarrhea. Although the birds most often do not show signs of disease, Salmonella
colonize the intestine and then systemically disseminate to the internal organs throughout the
body. As such, Salmonella can be found and spread to the environment via the feces; Salmonella
that infect the ovary and oviduct may contaminate the eggs before they are laid. Both S.
7
Enteritidis and S. Heidelberg are known for infecting the reproductive tract and being found in
eggs. Eggs that have been contaminated with Salmonella may have a higher level of embryo
mortality and death of newly hatched chicks [8].
Because birds can be infected with Salmonella without showing any signs, poultry
products have been identified as important sources of human foodborne illness. As of 2011 there
were an estimated 1.028 million cases of non-typhoidal salmonellosis with just over 19,000
hospitalizations and 452 deaths [17]. As of 2014, the top 6 serotypes were: Enteritidis,
Typhimurium, Newport, Javiana, I 4,[5],12:i:-, and Infantis [6]. In a report released by the CDC
[6], the top serovars for the year 2012 that were important for both human and poultry infection
were Enteritidis, Typhimurium, Heidelberg, Infantis, Montevideo, and I 4,[5],12:i:-.
Salmonella can be transmitted to birds through fomites such as people, water, feed, litter,
insects, rodents, wild birds, and many other sources. Fomites are any objects or substances that
can carry and transmit infectious organisms, such as vehicles, clothing, footwear, and equipment.
Insects and rodents can serve as reservoirs and vectors, spreading the disease from one flock to
another even when houses are cleaned out between flocks [10-21]; contaminated feed and water
are also important sources of infection [22]. Salmonella contamination can be introduced
anywhere along the production chain: in the hatchery, during transport, on the farm, in the
processing plant, or at home.
CONTROL OF SALMONELLA
There are many measures that can be taken to prevent Salmonella infection. These
include: maintaining Salmonella-free breeding stock; proper cleaning and disinfection of the
farm, hatchery, transportation crates, and equipment; strict biosecurity measures; the all in all out
8
system for flocks; feed additives and antibiotics; competitive exclusion; water sanitation; vector
and reservoir control such as insects and rodent control programs; and vaccination.
Vaccination is one biological measure that has been researched lately as a way to
decrease the potential of vertical transmission of Salmonella. Vaccination has been shown to be
effective in decreasing prevalence of S. Gallinarum, Entertidis, and Typhimurium. Vaccination
of hens may decrease the level of contamination, decreasing both vertical transmission as well as
egg shell contamination. This prevents colonization of chicks during hatching, and decreases the
prevalence of foodborne illness resulting from consumption of contaminated eggs. Additionally,
vaccination causes the formation of antibodies that can be passed from mother to egg, which can
also provide some protection against infection in newly hatched chicks [23-24]. Research has
shown that breeder flocks that had been vaccinated had progeny with lower prevalences and
loads of Salmonella than progeny from unvaccinated flocks [24-25]. Vaccination is more
effective for broiler breeders and for layers rather than broilers, because vaccinating broilers is
neither cost nor time effective. Vaccination can be used as part of a comprehensive prevention
program for controlling Salmonella in poultry. However, vaccines do not provide complete
protection or cross-protection against all serotypes [15].
Chicks do not have a fully developed immune system or intestinal microflora when they
first hatch; as such, they are highly susceptible to Salmonella. A developed intestinal microflora
makes it difficult for Salmonella to colonize and invade the intestinal tract, therefore limiting
pathogenicity. This can be accomplished in several ways: production of antimicrobial
substances; fewer niches for Salmonella colonization; stimulation of the immune system, and
competition for nutrients and other resources [15]. When one species outcompetes another for
the same resource, it is called competitive exclusion [26]. In an attempt to build a healthy
9
intestinal microflora in chicks, growers use feed additives, such as probiotics and prebiotics, so
as to increase resistance to Salmonella [22].
Preventive doses of antibiotics can also be given to prevent Salmonella infections but
antibiotic therapy is discouraged in food production animals because of the increase in antibiotic
resistance, including those serotypes that can cause human disease [27-28]. Giving antibiotics
prophylactically or as growth promoters may select for bacteria that are resistant, including
bacteria other than Salmonella, such as E. coli [29]. When those bacteria are transferred to
humans, such as through consumption, the genes for resistance to antibiotics can be transferred
to the normal microflora already present [28]. This poses a potential public health risk, as new
ways to treat antibiotic resistant bacteria and their diseases must be developed. Additionally,
there is public concern about residual antibiotic residues in meat, and most antibiotics are unable
to completely eliminate Salmonella, creating carrier birds that appear to be Salmonella negative
but are not [22]. Finally, there is a high cost to administer antibiotics, which gets passed on to the
customer in the form of more expensive products.
Cleaning followed by disinfection is one of the best ways to control the spread and
prevalence of Salmonella. Everything needs to be cleaned: the poultry houses between flocks
and all equipment inside, hatcheries and all equipment, processing plants and equipment, feed
mills, clothing, vehicles, and transport crates. Cleaning is defined as a mechanical process in
which organic matter is removed. Disinfection is defined as the process or agent used on
inanimate objects/surfaces that either destroys or renders inactive most microorganisms (but not
always spores) [30-31]. In order for disinfection to be effective cleaning must be done first.
Cleaning should include brushing, scraping, scrubbing, and dusting all of the litter,
debris, feces, feed, trash, debris, feathers, and removing any other organic matter from the
10
surfaces that will be disinfected. Equipment may need to be dismantled and cleaned separately; if
it can be cleaned well and efficiently inside the house, however, this is not necessary. The entire
house should then be pressure washed from ceiling to floor with hot water and a soap or
detergent. Rinsing after will remove any remaining matter and detergents, and decrease the
chances of animals coming into contact with residual detergents. After cleaning, there should not
be any visible organic matter left [8, 32]. Cleaning and disinfecting poultry houses between
flocks has been shown to reduce or eliminate infection [18, 33-35]. When cleaning and
disinfection is not done to a high standard, however, carry-over of Salmonella between
consecutive flocks is common [21, [33-35].
Maintaining cleanliness in transit as well as within the processing plant is also very
important. Efforts to reduce Salmonella in poultry carcasses during processing have an impact on
reducing human illness [36]. Heyndrickx et al. [37] found a correlation between Salmonella
positive fecal material in transport crates and contamination of carcasses. Transportation crates
are often contaminated with Salmonella [19, 38-39] and can therefore serve to spread the
bacteria.
DISINFECTION
After cleaning the house, all equipment should be disinfected. This can be done by
spraying or foaming all surfaces with a disinfectant, or by fogging. Concentrations and contact
times should be used according to the manufacturer recommendations. The type of disinfectant
selected depends on several factors: type of contaminating organism; degree of contamination;
presence of organic matter; contact time required for efficacy; toxicity to the environment;
application method; corrosiveness; type of surface to be treated; environmental factors such as
11
temperature, humidity, pH, and water hardness; stability during storage; and finally, cost [8, 40-
41]. Efficacy of disinfectants within a disinfectant group can also vary as a result of formulation
[20].
The efficacy of most disinfectants increases with temperature, and using higher
concentrations or longer contact times may compensate for temperatures that are lower than
recommended [41]. The temperature range in which a disinfectant is most effective varies. For
example glutaraldehyde based disinfectants are effective down to about 5 °C while formaldehyde
needs at least 16 °C.
Disinfectant manufacturers do research in order to determine the most effective
concentrations and contact times for their product, so anything less than recommended will not
have the desired effects and may even contribute to an increase in resistance by the
microorganisms [41]. Concentration also impacts the effectiveness of disinfectants. For example,
alcohol is most effective in the 60-90% range; peracetic acid, however, is effective at <0.3%
[31].
Another factor that affects disinfectant activity and efficacy is pH. The stability and
susceptibility of a bacterial membrane is partly due to electrical charges within its structure,
which can be altered by pH. Some types of disinfectants, such as oxidizing agents,
formaldehyde, and QACs, act by chemical reactions to molecules, compounds, structures, or
interrupting processes within the cell [42]. pH can impact the state of a disinfectant influencing
the ionization or by increasing/decreasing the concentration of the active compound. For
example, sodium hypochlorite ionizes to produce Na+ and the hypochlorite ion, OCl-. The OCl-
then establishes a pH dependent equilibrium with hypochlorous acid, HOCl. Phenols,
12
hypochlorites, and iodine may show decreased activity as pH increases; quaternary ammonium
compounds (QAC) and glutaraldehyde may show an increased activity [31].
One of the biggest factors that interferes with efficacy is the presence of organic matter
(OM). Organic matter can be in the form of feathers, litter, feces, carcasses, blood, eggs and egg
residues, chick fluff, etc. OM provides a protective physical barrier between disinfectants and
microorganisms, as well as providing habitat for bacteria to grow. This is why cleaning of a high
quality needs to be done before disinfection. The level of impact from OM depends on the
disinfectant. In a study by Stringfellow et al. [43] decreases in disinfectant efficacy were
observed in response to increasing levels of OM, dependent on dose. Phenolic disinfectants were
the least impacted and still reduced Staphylococcus aureus populations with the inclusion of 3%
OM, whereas a chlorhexidine compound was ineffective. OM may react with oxidative
disinfectants and neutralize others [41].
The efficacy of disinfectants is also dependent on the type, number and physiological
state of microorganisms. Salmonella are less tolerant to hypochlorite than Staphylococcus
aureus; S. Kentucky, S. ser. Montevideo, and S. ser. Senftenberg have been shown to be more
resistant to disinfectants than S. Typhimurium. If there is a high initial number of bacteria left
from cleaning, disinfection may not be completely effective and some cells may survive.
Bacteria that are actively growing are more susceptible to disinfectants than bacteria that are not
[41]. Bacteria that are in biofilms are more resistant [44-45]. We cannot completely control
which serotype may be present in the poultry house, and we can’t control what stage of life the
bacteria may be in but we can at least partly control the number of bacteria through a high
quality cleaning before disinfection.
13
Bacterial resistance is becoming a bigger health issue every year; as Salmonella and other
microorganisms become resistant to disinfectants and antibiotics, the severity of illness increases
and the probability of full recovery decreases because there are fewer options for prevention and
treatment. Bacterial resistance varies between different types and species of microorganisms;
within Salmonella, resistance also varies between serotypes. For example; Salmonella strains
Agona, Kentucky, Montevideo and Senftenberg are more resistant to disinfectants as compared
to S. Typhimurium ATCC 13311 [41]. One of the biggest drivers of bacterial resistance is using
biocides, antibiotics, and disinfectants at sub-lethal levels. A study looked at susceptibility to
chlorhexidine in S. Typhimurium and found that sub-lethal concentrations of the chemical was
capable of inducing physiological changes that decreased susceptibility [46].
There is evidence that building material may have an effect on efficacy of disinfectants
[44-45, 47]. Berchieri and Barrow [47] found that wood shavings neutralized some of the
sanitizers used, whereas metal foil and polythene strips did not have an effect. Wood is hard to
clean due to its structure and nature. The variable surface is conducive for biofilm formation
while cracks and crevices provide protection for bacteria from cleaning and disinfectants. Using
metal, plastics, or other materials to either replace or cover wood surfaces in poultry housing is
likely to improve both ease and efficacy of cleaning and disinfection.
In broiler houses the most commonly used classes of disinfectants are: peroxides and
oxidizing agents, alcohols, halogens, QACs, aldehydes, phenolic compounds, and biguanides[22,
43]
14
CLASSES OF DISINFECTANTS
Alcohols
Alcohols are effective against vegetative bacteria, viruses, and fungi, but are not
sporicidal. They have rapid broad-spectrum antimicrobial activity and are often used for
disinfecting hard surfaces and skin as they leave no residue. They are often used in formulations
combined with other disinfectants [48]. Ethyl alcohol and isopropyl alcohol are the most widely
used alcohols, and isopropyl alcohol is considered slightly more effective against bacteria.
Alcohols are most effective in concentrations between 60-90%, and efficacy significantly drops
when concentration is below 50%. An ethanol concentration of 70% is effective against
Salmonella on surfaces, including Salmonella in biofilms found in the feed industry [49].
Efficacy is increased in the presence of water, and many alcohol products have low levels of
other biocides mixed in. These other products can increase product efficacy by decreasing the
evaporation time of the alcohol or remain on the skin following the evaporation and have their
own biocidal activity [31]. However, alcohols are good for situations where low residual water
after use is necessary because of their quick evaporation time [49].
Alcohols are not effective in the presence of organic matter, and are expensive so use is
mainly restricted to hand sanitizers and equipment that cannot be cleaned with water. They are
also very flammable and can pose a safety and health risk [40-41].
Aldehydes
Aldehydes are organic compounds that include formaldehyde and glutaraldehyde.
Aldehydes are potentially carcinogenic and are irritating and toxic to humans. They are effective
in the presence of organic matter, act rather slowly, reactive with other chemicals, and are
potentially dangerous to respiratory systems [48].
15
Formaldehyde exists naturally as a gas but is used as an aqueous solution with 34-38%
formaldehyde with methanol called formalin. Formaldehyde is bactericidal, sporicidal, and
virucidal. It is very reactive, interacting with protein, DNA, and RNA [31]. Formaldehyde is
very potent but is also highly toxic to humans and animals so must be used with caution in a well
ventilated area [40]. Because formaldehyde is so toxic and needs to be handled carefully it is best
applied by specialist contractors, which may incur extra costs [34]. Formaldehyde has shown
efficacy against Salmonella in the presence of organic matter [50].
Glutaraldehyde also has a broad spectrum of activity against bacteria, spores, fungi, and
viruses. It works more quickly than formaldehyde and is also effective in the presence of OM
[47]. Glutaraldehyde associates strongly with the outer layers of bacteria, inhibits transport and
enzyme systems, and inhibits RNA, DNA, and protein synthesis. It is more active at alkaline pHs
[31].
Oxidizing Agents
Oxidizing agents produce free radicals that act by disrupting lipid membranes, proteins,
and nucleic acids. The most common oxidizing agents are hydrogen peroxide and peracetic acid
[31, 50]. Oxidizing agents have moderate to wide germicidal activity, are moderately corrosive,
and not very toxic. They have little residual activity and limited shelf life [40]. They have some
activity in the presence of OM, although this seems to be debated[51-52].
Hydrogen peroxide (H2O2) has broad-spectrum efficacy against viruses, bacteria, yeasts,
and bacterial spores. It tends to have better activity against gram-positive bacteria than gram-
negative bacteria. Hydrogen peroxide can be bought in concentrations ranging from 3-90%. and
16
is considered environmentally friendly because it breaks down into water and oxygen [31].
H2O2 is reactive and is not very stable [48].
Peracetic acid is a mix of hydrogen peroxide and acetic acid. It also decomposes into safe
by-products, acetic acid and oxygen, but is considered more potent than H2O2. It is also more
likely to remain active in OM than H2O2 [48, 51].
Acetic acid, more commonly known as vinegar, can inhibit carbohydrate metabolism and
can induce apoptosis, resulting in the death of an organism. Acetic acid up to 2.5% is allowed as
a processing aid on carcass surfaces in the US [53]. A study by Nei et. al [54] found that a
gaseous acetic acid treatment on black pepper and fenugreek seeds produced an approximate 5.0
log CFU/g reduction in Salmonella Enteritidis after 3 hours. Reduction of S. Enteritidis in that
study after only one hour was still significant, showing that a gaseous application of acetic acid
can be one option for disinfection. A study by Chang and Fang [55] found a 3 log reduction of
E.coli on inoculated lettuce that had been exposed to commercial vinegar containing 5% acetic
acid for 5 minutes. Although E. coli and Salmonella are not the same, they are similar and it is
reasonable to believe that they have similar susceptibilities to disinfectants.
Citric acid is naturally found in citrus fruits and is another organic acid that can
potentially be used as a disinfectant. A study by Sengun and Karapinar [56] using fresh lemon
juice to disinfect carrots found log reductions of S. Typhimurium ranging from 0.079-3.95,
depending on time and rate of bacterial inoculation. The times tested were 0 minutes, 15, 30, and
60 minutes. The study also tested the efficacy of mixing acetic and citric acid together and found
the mixture to be more effective than either acid alone.
17
Quaternary Ammonium Compounds
Quaternary ammonium compounds (QAC) are surfactant compounds made of a
hydrophobic group and a cationic group. QACs are excellent for hard-surface cleaning and
deodorization. They work by interacting with phospholipid components and causing generalized
damage to the membrane [31]. QACs are not effective against non-enveloped viruses, fungi, and
bacterial spores, but are effective against gram positive and negative bacteria and enveloped
viruses. QACs are more effective in preventing bacterial growth, rather than killing them, and
they tend to be more active against Gram-positive bacteria rather than Gram-negative [48]. Hard
water, other soaps/detergents, and OM limits efficacy; however, QACs are reasonably quick
acting, have a low cost, and are relatively noncorrosive and nontoxic [40, 48, 57].
Phenols
Phenols are substances derived from coal tar and have been used for many years [48].
They act by inducing progressive leakage of intracellular components. Phenolic disinfectants are
still effective in OM [43, 47-48]. They have a broad spectrum of activity, are effective in hard
water, are of low to moderate cost, and have little residual activity unless mixed with other
compounds [40]. However, efficacy, toxicity, and corrosiveness greatly varies between
formulations [43, 48, 50, 58].
Biguanides
Biguanides are organic compounds that feature a specific formula and functional group.
The most commonly used biguanide is chlorhexidine. Chlorhexidine is bactericidal but is not
sporicidal and has limited antiviral properties. Its activity is pH dependent and can be greatly
18
reduced when there is OM present [43]. Chlorhexidine has a two-phase effect: in the first, it
works by damaging the cell membrane. If concentrations of chlorhexidine are high, after the
membrane is damaged the intracellular components become coagulated [31]. Biguanides have
good residual activity and are relatively nonirritating to tissues, nontoxic, and cheap [5, 40].
Halogens
Halogens are derived from the halogen group of the periodic table. Chlorine and iodine
based compounds are the most common halogens used as disinfectants.
Chlorine Releasing Compounds. Chlorine disinfectants are active against viruses, fungi,
algae, and bacteria. They are not effective against spores. They corrode metals, deteriorate
fabrics, and can cause irritation in high concentrations. They are cheap and active in low
concentrations but inactivated by OM [40]. Although the mechanism of action for chlorine
disinfectants is not known, it is known that they are oxidizing agents. Therefore, chlorine
disinfectants are most active between pH 4 and 7 when there is the most of HClO, the compound
that actively oxidizes. Chlorine disinfectants disrupt protein activity as well as inhibit DNA
synthesis [31].
Bleach, or sodium hypochlorite, is a common example. Bleach is commonly used in the
food industry, especially for washing produce. Sodium hypochlorite is used for more than
washing produce, however, as it has also been to treat water, deodorize, stain removal, and
disinfect equipment for various uses. Bleach is very effective against all types of
microorganisms, is easy to use, and relatively cheap. However, concentrated solutions are
corrosive to skin, metals, and other materials, and sodium hypochlorite tends to break down over
time [48].
19
Another common and important chlorine releasing agent is chlorine dioxide, which is
also used for food processing as well as routinely utilized in the process for disinfection of
drinking water.
Iodine and Iodophors. Iodine based disinfectants have been used for many years and are
less reactive than chlorine but rapidly bactericidal, fungicidal, virucidal, and sporicidal at both
low and high temperatures. Because aqueous and alcoholic solutions of iodine cause staining and
are generally unstable, iodophors, complexes of iodine and a solubilizing agent or carrier, have
been developed [31, 48]. Some of the iodophors have been developed to minimize staining and
irritancy. Iodine based disinfectants act upon proteins, nucleotides, and fatty acids. Iodine and
iodophors become inactivated in the presence of OM [47].
INTERNAL PARASITES
Despite being a very important disease, Salmonella is not the only organism that causes
infection and disease in poultry. Both commercial and backyard flocks of all sizes can be
plagued by internal parasites; some of the most important include members of the genera
Ascaridia, Eimeria, Capillaria, and Heterakis.
Ascaridia
Members of the genus Ascaridia are round worms that are commonly found in poultry.
They are usually in the small intestine but birds with heavy infestations can have worms in other
parts of the digestive system, as well as in the reproductive system. If the round worms become
established in the reproductive system, occasionally worms may be found in eggs laid by the
hens. Adult worms range from 2 -4.5 inches, with the females being larger than the males. They
are thick and yellow-white worms.
20
The life cycle of round worms is direct – there is no intermediate host. Eggs are passed in
the feces of the host and develop outside of the bird in about 10-12 days under ideal conditions;
longer if conditions are less favorable. Eggs are resistant to low temperatures and can remain
viable for over 3 months. However, eggs are easily killed by hot, dry weather. Poultry become
infected when they swallow the eggs with food or water, or ingest earthworms that swallow the
eggs. Once swallowed by the host, the eggs will hatch in the intestine. The larva will go through
several stages before maturing in 28-30 days after hatch.
Infection by Ascaridia can cause weight depression, weight loss, and decrease in egg
production. Heavy infections of round worms can actually obstruct the intestine. Other
symptoms include anemia, diarrhea, retarded growth, and lesions in the intestine. Birds younger
than 3 months are more susceptible to infection, as well as birds with dietary deficiencies such as
protein or vitamins A and B [8].
In addition to the symptoms of ascaridiasis, a correlation with Salmonella infection has
been found. The eggs of A. galli can transfer Salmonella and therefore increase the risk of
persistence in the environment[59]. Additionally, a study by Eigaard et al. [60] found that
infection with A. galli may increase the colonization rate of S. Enteritidis and prolong the
shedding of bacteria in the feces, leading to increased risk of infection in other birds.
Birds that are infected with ascarids can be treated with peperazine products, which are
relatively nontoxic to poultry.
21
Heterakis gallinarum
These are worms that are found in the ceca of poultry. These are small worms, ranging in
size from 0.25-0.6 in long. The females are larger than the males.
Cecal worm eggs are passed out in the feces of the host and develop outside the body.
Under optimal conditions the eggs become infective after 2 weeks. The eggs are very resistant
and may remain viable for years. Once ingested, infective eggs hatch in the intestine and within
24 hours may be found in the ceca, where they go through several stages and molts. Cecal worms
mature 24-30 days after hatch [8].
Even chickens with heavy infections show very few signs of infection, other than
inflammation and thickening of the ceca walls in necropsy. The importance of H. gallinarum lies
in its role as a carrier for the disease in turkeys known as blackhead, caused by Histomonas
meleagridis. H. meleagridis can be found in both the eggs and the bodies of cecal worms.
Medications that can be used to treat against cecal worms include fenbendazole,
Hygromycin B, and ivermectin. Ivermectin is not approved for use in poultry, however.
Capillaria
Species of the genus Capillaria are known as hair, crop, and/or thread worms. There are
6 species that are commonly found in poultry, are small and thin, resembling hair or thread, and
range in size from 0.20-3.1 inches long, depending on species.
Two species, C. annulata and C. contorta, are found in the crop and esophagus. Three
species are found in the small intestine, and the final species, C. anatis, occurs in the ceca. For
some species, the life cycle is direct; eggs shed in the feces become infective, are consumed, and
cause infection. Eggs voided in the feces usually take 9-15 days to become infective, then mature
22
after 18-60 days after hatching in the host, depending on species and conditions. In the other
species the life cycle requires an earthworm as an intermediate host. The eggs are shed from the
host and must be swallowed by earthworms before becoming infective. Birds then consume the
earthworms to become infected. The eggs become infective 22-25 days after leaving the host
and hatched worms mature after 20-26 days.
Crop worms cause inflammation and thickening of the crop and esophagus. In heavy
infections, the crop may become dysfunctional. Hair worms can cause diarrhea and inflammation
and thickening of the intestine. Other symptoms from infection by any of the species include
depression, malnutrition, emaciation, anemia, and death [8].
Medications that can be used to treat Capillaria infections include methyridine and
fenbendazole. Other compounds that are effective but not approved for use in poultry include
ivermectin, tetramisole, flubendazole, phenothiazine, and haloxon.
Eimeria
Coccidiosis is caused by a protozoan parasite of the genus Eimeria. There are 7 species
that cause disease in chickens and they live and multiply in the cells of the intestinal tract,
including the ceca.
Birds become infected after consuming infective oocysts, thick-walled structures
containing the parasite. Once ingested oocysts break down and release the sporozoites, a stage of
the parasite, that then enter the cells lining the intestines. The sporozoites multiply and rupture
the cells in order to escape. Sporozoites may go through multiple stages and cycles of entering
and rupturing cells before forming oocysts that are released in the feces. Oocysts require 12-48
hours to become infective, depending on species and conditions such as temperature and
23
humidity. The minimum time between ingestion and new oocysts being released, the prepatent
period, ranges between 4-6 days.
Signs of coccidiosis may include decreased feed and water consumption, decreased egg
production, pigmentation loss, weight loss, slow growth and poor feed conversion, bloody
diarrhea, and high mortality. Coccidiosis affects younger birds usually 3-6 weeks of age before
they develop immunity; however, it can affect older birds. The severity of infection depends on
the health of the bird and the number of oocysts ingested. Chickens will usually develop
immunity quickly, thus self-limiting the infection. Chickens may be infected with multiple
species of coccidia at the same time, and immunity to one species will not prevent infection with
another species [8].
Eimeria has additional importance because infection with coccidia has been shown to
enhance establishment and persistence of S. Typhimurium infection [61-64]. It has also been
shown that coccidial infection can induce a recrudescence of previous Salmonella infection [65].
Coccidia are commonly found everywhere, so it is not possible to completely eliminate or
prevent infection through quarantine, disinfection or sanitation. Oocysts are extremely resistant
to common disinfectants, and it is not possible to completely sterilize a chicken house. There are
numerous anticoccidials available on the market, and few are effective against all species of
coccidia. Medications that can be used to treat a coccidiosis infection include quinolones,
sulfonamides, and ionophores.
24
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30
CHAPTER 2
Comparisons of selected household and commercial disinfectants against poultry
Salmonella isolates
31
Comparisons of selected household and commercial disinfectants against poultry
Salmonella isolates
K. Moyle*1, F.D. Clark*, and J.R. Moyle†
*University of Arkansas, Division of Agriculture, Center of Excellence for Poultry Science,
Fayetteville, AR
†University of Maryland Extension, College Park, MD
Primary Audience: Small and Medium-Scale Poultry Producers, Organic and Sustainable
Agricultural Practitioners, International Poultry Producers
SUMMARY
Salmonella is one of the most important bacteria to control in poultry as it not only
impacts poultry production but also poses a threat to public health. Disinfection of housing,
materials, equipment, etc., after cleaning is one of the best ways to protect against infection.
There are several different classes of disinfectants and within those classes, numerous
formulations. Additionally, there is a ‘green’ or ‘organic’ movement in the general populace
away from conventional chemicals towards items that are seen as more natural. The preparation,
use, and efficacy of each disinfectant vary, so the objective of this study was to determine the
efficacy of 3 commercial disinfectants at various dilutions over the course of 4 time periods. Six
serotypes of Salmonella were tested. In addition to the commercial disinfectants, lemon juice,
bleach, and vinegar were also tested for efficacy at various dilutions over the course of 4 time
points. Results showed bleach to be very effective, followed by the commercial disinfectants.
Efficacy is dependent on serotype, concentration of disinfectant, and contact time.
1 Corresponding author: [email protected]
32
Key words: Salmonella, disinfectant, poultry
DESCRIPTION OF PROBLEM
Salmonella are a gram-negative bacterium that can be found all over the world. They can
cause disease in animals and humans, and is naturally zoonotic[1-2]; therefore, control is
essential. Because of this demand, many varieties of chemical disinfectants have been developed,
each with varying properties and abilities[3-5]. Recent outbreaks of Salmonellosis caused by
interaction with backyard flocks[6] have shown that health of small flocks is as important as the
health of commercial flocks. Although there are various disinfectants available in developed
areas for control of Salmonella, people in rural and developing areas may not always have access
to such. There has also been a movement in developed areas away from chemical disinfectants,
towards what is seen by the general populace as more natural or organic compounds and
methods of disinfection. Due to interest in alternatives and unavailability for others, this project
looks at the efficacy of three commercial disinfectants (Virkon-S, Virocid, and Pheno-Tek) and
three common household compounds (lemon juice, bleach, and vinegar) that have some
antibacterial properties and are often readily available, even in rural and developing areas. All six
disinfectants were tested at varying dilutions against six serotypes of Salmonella over the course
of four time points. Bleach was found to be very effective, with rapid action and maintaining
effectiveness at low concentrations. The commercial disinfectants were more effective than
lemon juice and vinegar.
33
MATERIALS AND METHODS
Salmonella Isolates
Six Salmonella isolates were obtained from Cobb Vantress Laboratory, isolated from
breeder flocks. The six isolates were: Salmonella Enteritidis, Kentucky, Senftenberg,
Montevideo, Typhimurium, and Heidelberg.
Each isolate was maintained in a frozen glycerol stock. The glycerol stock was made by
growing isolates in Brain Heart Infusion (BHI) broth for 24 hours. An aliquot of the broth was
then centrifuged; the supernatant was discarded. The pellet was reconstituted with a 20%
glycerol and BHI mix. An aliquot was then dispensed in cryovials and stored in a -70 degree
ultralow.
A loop full of glycerol stock was mixed with 5 ml of BHI and incubated for 24 hours
before testing with disinfectants.
Agar Plate Preparation
Nutrient agar plates were prepared by mixing 23 grams of powdered DIFCO nutrient agar
with one liter of distilled water in a glass beaker. The solution was mixed on a heated stirring
plate for 25-30 minutes then boiled for one minute to completely dissolve the powder. After
autoclaving for 15 minutes, fifteen ml of the mixture was pipetted into a 100 x 15mm plastic
petri dish. Two to three prepared petri dishes were placed in a bacteriological incubator and
incubated at 37°C for 24 hours. The plates were checked after 24 hours of incubation for
contaminants.
34
Disinfectants
Disinfectant solutions were prepared at 4 concentrations. Common household items were
lemon juice (ReaLemon 100% lemon juice), bleach (Great Value), and distilled white vinegar
(Great Value). Each were tested at full strength (straight from the bottle), half strength, quarter
strength, and 1/10 strength. Bleach was additionally serially diluted to 1/100, 1/500, 1/1000, and
1/5000 concentrations.
Commercial disinfectants were used at standard concentration, half concentration, 1/10
concentration, and 1/100 concentration. The directions for making the standard concentration of
the commercial disinfectants were on the label of each disinfectant.
The standard concentration of Virkon-S was mixed in a 500 ml Pyrex bottle. One tablet
(0.18 oz.) was mixed with one pint of distilled water, according to directions on the label.
The standard concentration of Virocid was prepared in a 50 ml centrifuge tube by mixing
0.1 ml of disinfectant with 40 ml of distilled water.
The standard concentration of Pheno-Tek was prepared in a 50 ml centrifuge tube by
mixing 0.1 ml with 25.6 ml of distilled water.
The 0.5X concentrations of all disinfectants used was created by mixing 2 ml of standard
concentration with 2 ml of PBS. The 0.1X concentration of all disinfectants was created by
adding .5 ml of standard concentration to 4.5 ml of PBS. The 0.25X concentration of the
household items was created by mixing 1 ml of the disinfectant with 3 ml of PBS. The 0.01
concentration of the commercial disinfectants was created by mixing 0.05 ml of standard
disinfectant with 4.95 ml of PBS. All concentrations were created in 14 ml culture tubes.
35
Preparation of Bacterial Solutions
To prepare the bacterial solution, a loopful of the frozen bacteria glycerol stock was
mixed with 5 ml of BHI in a 14 ml tube then placed in the incubator for 24 hours. The next day,
the solution was vortexed and the optical density was determined.
The optical density of the bacterial solution was determined using a spectrophotometer at
625nm wavelength by mixing BHI and bacterial solution in a cuvette tube. The target Optical
Density (OD) value was between 0.6 and 0.8. If an OD reading was outside of the range then
BHI was added to the cuvette to dilute the solution and another reading was taken. This is the
bacterial solution that was used to test the efficacy of disinfectants. A tube containing only BHI
was used as a blank for a control.
The optical densities of the Salmonella solutions were determined to estimate the
concentration of bacterial cells in suspension. The target estimation of cells was 4 × 108 to 6 ×
108 colony forming units (CFU)/ml, which is equivalent to an optical density of 0.6-0.8, based on
a growth curve for S. Enteritidis [7]. However, since this was only an estimate a plate count was
performed for each isolate to determine the actual number of bacterial cells in the solution at the
time of testing.
Standard Plate Count
A standard plate count was performed after obtaining the optical density range for each
Salmonella isolate. The standard plate count method was used to determine the number of viable
bacterial cells per unit volume of a sample using nutrient agar plates.
In a culture tube (12 x 75mm 5 ml), 0.1 ml of bacterial solution was added to 0.9 ml PBS
and vortexed to make 1/10 dilution. A 0.1 ml aliquot from this tube was transferred to another
36
tube also containing 0.9 ml of PBS and vortexed to make the 1/100 dilution. This procedure was
repeated until the 10-10 dilution was achieved. The tubes having serial solutions of 10-5 to 10-10
were plated in triplicate on nutrient agar plates to count the bacteria. A 0.1 ml aliquot from each
tube was plated onto nutrient agar plates and evenly distributed on the plate with the help of an L
shaped glass rod. The plates were placed in an incubator at 37 °C for 24 hours. After 24 hours,
the colonies on the plates were counted and recorded.
Efficacy of Disinfectant Against Salmonella Isolates
After the optical density has been determined and the standard plate count started, 0.1 ml
aliquots of a bacterial solution were placed in 1.7 ml microcentrifuge tubes and centrifuged at
13,000 rpm for 5 minutes to pelletize the bacteria. The supernatant was discarded and the pellet
was resuspended with 1.5 ml of a disinfectant solution. Timing began as soon as a disinfectant
solution was added to the tube. After 5, 15, 30, and 60 minutes 0.1 ml from the tube was plated
on nutrient agar plates with the help of an L-shaped glass rod. Three replicate plates were
prepared for each time. After inoculating the solution on each plate, the L shaped glass rod was
dipped in 70% alcohol and heated with a flame to sterilize it. After incubation for 24 hours, the
colonies on the plates were counted and recorded.
RESULTS AND DISCUSSION
Efficacy of Disinfectants Against S. Enteritidis
Full strength lemon juice reduced the bacterial concentration by 5.84 logs after only 5
minutes, and after 15 minutes no colonies were isolated. Half strength lemon juice reduced
bacterial counts by 7.03 logs after 15 minutes and killed all by 30 minutes. Quarter strength juice
37
also killed after 30 minutes, but 0.1 strength lemon juice did not reduce bacterial counts until an
hour after contact.
Full strength and half strength vinegar were able to completely kill S. Enteritidis after
only 5 minutes, and colonies were never isolated from any following time point. Quarter strength
vinegar reduced bacterial concentration by 4.97 logs after 15 and 30 minutes, and killed by an
hour after contact. 0.1 strength vinegar was not effective at any tested time point.
Virkon-S performed similarly to vinegar in this instance; no bacteria were isolated from
the X and 0.5X concentrations of Virkon at any time point. The 0.1X concentration reduced
bacterial counts by 4.88 logs after 15 minutes and 6.83 logs after 30 minutes. There were no
colonies observed after one hour. The 0.01X concentration was not effective.
Virocid was effective at the X and 0.5X concentrations; no bacteria were observed at any
time. However, the 0.1X concentration was only effective after one hour and the 0.01
concentration was not effective at any time.
Pheno-Tek killed at all time points with the X and 0.5X concentrations. It did not show
evidence of effectively reducing or killing at any time when diluted to 0.1X or 0.01X.
Bleach was effective at killing at all time points at all concentrations measuring between
X to 0.01X. The 0.002X concentration was not effective within 5 minutes of contact but killed at
all later time points. The 0.001X dilution was not effective within 5 minutes either, but reduced
by 6.28 logs after 15 minutes and 6.34 after 30 minutes. No colonies were observed after one
hour. The 0.0002X concentration was never effective (Table 2).
38
Efficacy of Disinfectants Against S. Heidelberg
Full strength lemon juice was not effective after 5 minutes, but after 15 minutes no
colonies were observed. The 0.5X concentration reduced bacterial count by 6.76 logs after 30
minutes and killed within 60 minutes. The 0.25X concentration didn’t reduce bacterial count
until 60 minutes, and the 0.1X concentration was not effective at any time.
Vinegar reduced bacterial counts by 6.63 logs after 15 minutes with the full strength
concentration, and killed after 30 minutes. The 0.5X concentration reduced bacteria by 6.98 logs
after 30 minutes and killed after 60. The 0.25X and 0.1X concentrations were not effective at any
time.
Virkon-S and Virocid both performed similarly with this strain of bacteria. The X and
0.5X concentrations killed at all time points. Bacterial counts were reduced by 6.16 logs after 15
minutes with the 0.1X concentration of Virocid and killed thereafter. 0.1 Virkon-S reduced
counts by 5.39 logs. The 0.01X concentration of either disinfectant was not effective at any time.
No colonies were observed with the X and 0.5X concentrations of Pheno-Tek at any
time; however, neither the 0.1X nor the 0.01X concentrations were effective at any time.
Bleach was effective at killing at all time points at all concentrations measuring between
X and 0.002X. S. Heidelberg was affected differently by the 0.001X concentration. After 5
minutes, no bacteria were observed; however, too many colonies to count were observed after 15
minutes. There was a reduction in colonies again after 30 minutes, and after an hour no colonies
were isolated. The 0.0002X concentration of bleach was not effective (Table 3).
39
Efficacy of Disinfectants Against S. Kentucky
Lemon juice at full strength was efficacious after 15 minutes, killing all bacteria. No
bacterial colonies were observed after 30 minutes with the 0.5X solution. The 0.25X
concentration reduced bacterial counts by 6.33 logs after 30 minutes and killed after one hour,
while the 0.1X concentration was not effective at any time.
Full strength vinegar was effective at all times; half strength vinegar reduced counts 5.98
logs after 5 minutes and killed at the following time points. The 0.25X concentration didn’t
reduce counts until 30 minutes (7.17 logs) but killed after 1 hour, and the 0.1X concentration was
not effective at any time.
Virkon-S was effective at killing S. Kentucky at all time points at both full strength and
half strength. The 0.1X concentration reduced bacterial counts 5.45 log after 30 minutes and
killed after one hour. The 0.01X concentration did not reduce bacterial counts for any time point
tested.
Virocid was also effective at killing S. Kentucky at all time points at both full strength
and half strength. After 15 minutes there were no colonies observed with the 0.1X concentration;
however, there were colonies observed after 30 minutes but not after one hour. It is possible that
there was contamination somehow. The 0.01X concentration did not reduce or kill the bacteria
present.
Pheno-Tek killed all bacteria present at all times with both the X and 0.5X
concentrations. The 0.1X concentration did not reduce the bacterial count until the hour time
40
point (5 log reduction), and the 0.01X concentration never effectively reduced the bacteria
present.
Bleach was effective at killing at all time points at all concentrations measuring between
X to 0.001X. The 0.0002X dilution was not effective after 5 minutes but did decrease the
bacteria by 5.21 logs after 15 minutes. No bacteria were recovered at 30 or 60 minutes (Table 4).
Efficacy of Disinfectants Against S. Typhimurium
Lemon juice at full strength was not effective at 5 minutes, but at 15 minutes and after no
colonies were observed. Colonies were reduced by 7.72 logs after 15 minutes with the .5X juice
and killed after 30. The 0.25X were not effective at 5 or 15 minutes but killed at 30 and one
hour. The 0.1X lemon juice reduced bacterial counts 8.28 log after one hour but were ineffective
until then.
Full strength vinegar was efficacious in killing at all times, and 0.5X was able to reduce the
bacteria count 7.61 log after 5 minutes and kill it thereafter. The 0.25X vinegar reduced counts
8.16 log after 15 minutes and killed at the following time points, whereas the 0.1X vinegar was
never effective.
Virkon-S was effective at killing at all measured times at both the X and 0.5X
concentrations. The 0.1X concentration did not reduce counts until 30 minutes (7.2 log), killing
by one hour. The 0.01X solution was never effective.
There were no bacterial colonies observed at any time for both the X and 0.5X
concentrations of Virocid. The 0.1X concentration reduced counts 6.37 log after 5 minutes,
reduced by 7.35 after 15 minutes, and killed after 30; however, the 0.01X solution never reduced
counts successfully.
41
Pheno-Tek was completely effective with both the X and 0.5X solutions; it was
completely ineffective with both the 0.1X and 0.01X concentrations.
Bleach was effective at killing at all time points at all concentrations measuring between
X to 0.002X. The .0.001X concentration caused a 6.76 log reduction and killed thereafter. The
0.0002X dilution was not effective at 5, 15, or 30 minutes, but no bacteria were recovered after
one hour (Table 5).
Efficacy of Disinfectants Against S. Senftenberg
No colonies were ever observed for full strength lemon juice. Bacterial counts for the half
strength juice were reduced 7.35 logs after 15 minutes, and none were observed for time points
after. The 0.25X solution was not effective at 5 or 15 minutes, but it killed at 30 and 60 minutes.
The 0.1X concentration was able to reduce the bacterial count by 6.18 logs only after an hour
had passed.
Vinegar was effective at killing at both full strength and half strength. However, the
quarter strength solution didn’t reduce counts (6.17 logs) until 15 minutes, and killed all at 30
and 60 minutes. The 0.1X concentration was never effective.
Virkon-S and Virocid performed similarly when tested against S. Senftenberg. Both the X
and 0.5X solutions killed the bacteria at all time points, and the 0.1X Virkon-S reduced count
4.89 logs after 15 minutes. 0.1X Virocid reduced by 6.6 logs. No colonies were found after 30
and 60 minutes, and the 0.01X solution was completely ineffective for all times.
Pheno-Tek was effective at full strength and half strength at all times, and ineffective at
0.1X and 0.01X concentrations for all times.
42
Bleach was effective at killing at all time points at all concentrations measuring between
X and 0.002X. The 0.001X solution reduced bacterial counts after 5 minutes and killed after; the
0.0002X concentration was ineffective (Table 6).
Efficacy of Disinfectants Against S. Montevideo
Lemon juice at full strength was not effective at 5 minutes, but killed S. Montevideo at
the later time points. The 0.5X solution reduced by 5.49 logs after 15 minutes and killed at 30
and 60 minutes, while the 0.25X solution reduced by 6.9 logs at 30 minutes and killed after one
hour. The 0.1X solution was ineffective until it reduced bacterial counts 5.82 logs after an hour.
Vinegar was slightly more effective against S. Montevideo than was lemon juice. Both
the X and 0.5X solutions killed all bacteria for all times, while the 0.25X solution killed all
bacteria at 30 and 60 minutes. Unlike lemon juice, however, the 0.1X concentration of vinegar
was not effective at reducing or killing the bacteria at any time.
Virkon-S killed at all times with the full and half strength concentrations. The 0.1X
concentration was able to reduce the bacterial count 7.6 log and 8.77 log after 30 and 60 minutes,
respectively, while the 0.01 concentration was completely ineffective.
Once again, there were no colonies found for any time point with the full and half
strength solutions of Virocid. The 0.1 concentration was ineffective until it reduced counts 7.82
log after one hour, and the 0.01 concentration never reduced counts.
Pheno-Tek was effective at full strength and half strength at all times, and ineffective at
0.1X and 0.01X concentrations for all times.
43
Bleach was effective at killing at all time points at all concentrations measuring between
X to 0.01X. There were reductions in the number of colonies observed after 5 and 15 minutes
with the 0.002X strength solution, and no colonies recovered at later times. S. Montevideo also
had an interesting response to the 0.001X concentration; colony counts were reduced after 5
minutes, but were too numerous to count after 15 minutes. At 30 and 60 minutes, however, no
colonies were isolated. As similar to the other serotypes, the 0.0002X solution was not
efficacious (Table 7).
Number of Salmonella Strains Each Disinfectant Reduced/Killed at Each Time
Out of all the disinfectants, bleach was by far the most effective since it reduced/killed all
six Salmonella serotypes within 5 minutes at the 0.01X concentration. The three commercial
disinfectants (Virkon-S, Virocid, Pheno-Tek) were completely ineffective at that low of a
concentration and the two common household disinfectants (vinegar, lemon juice) were not
effective at the 0.1X.
The commercial disinfectants were equal at the full and half strength concentrations;
variation was seen at the 0.1X solution. Virkon-S killed fewer serotypes than Virocid after 15
minutes, but then killed more after 30 minutes. Pheno-Tek was ineffective at the 0.1X and 0.01X
concentrations.
Full strength vinegar affected more serotypes after 5 minutes than lemon juice; they were
equal after 15 minutes. At the 0.5X and 0.25X concentrations vinegar was more effective than
lemon juice at all times. However, 0.1X lemon juice retained some efficacy after an hour
whereas vinegar was not effective at that concentration, regardless of time (Table 8).
44
Discussion
One important lesson demonstrated in this experiment is that serotype matters when
attempting to control Salmonella. Even though the various strains all belong to the same species,
they react differently to various disinfectants. For example, lemon juice and vinegar are much
more effective against S. Senftenberg than against S. Heidelberg; however, they both react
similarly to Pheno-Tek. S. Kentucky and S. Montevideo were still present after 30 minutes of
exposure to 0.1X Virkon-S, while S. Heidelberg and Senftenberg were not. If farmers know what
serotype of Salmonella they have present, they can choose a disinfectant that will be more
efficient and effective.
A comparison to similar research done by Aslam [7] indicates that bacteria can become
resistant to disinfectants within a few years. Some of the strains of Salmonella used in this
experiment were also used in the previous research, and results suggest that within that time
frame the bacteria adapted. In the research by Aslam, S. Kentucky was killed by 0.1X
concentration of Virkon-S by 30 minutes, whereas present data indicates the same concentration
didn’t reduce bacterial counts until 30 minutes had passed and did not kill all bacteria present
until an hour after contact. Virocid at 0.1X concentration killed all S. Enteritidis by his 10 min
time point, whereas current data indicates Virocid didn’t kill until 1 hour after contact. One
plausible explanation for this discrepancy between studies would be that the bacteria have
evolved and developed a resistance to certain disinfectants in the time period between
experiments.
45
Disinfectants can be effective at lower concentrations when contact time is increased. For
example, Virocid at 0.1X was not effective at 5 minutes but grew increasingly more so at later
time points. All strains but S. Montevideo were killed with 0.1X by one hour after contact.
It is reasonable to assume that some people using disinfectants will dilute products in
order to save costs; this can be effective but has limitations. In this study, some disinfectants
perform just as well at half the recommended concentration as at the recommended
concentration. However, performance at further dilutions decreases and eventually becomes
completely ineffective, regardless of contact time. Rather than diluting commercial disinfectants
to cut costs, it is possible to consider an alternative such as bleach, which is low cost and highly
effective.
Many hobby farmers routinely use apple cider vinegar in the water of the birds to help
maintain health and fight disease (F. D. Clark, personal communication, 2016). This supports the
idea of using vinegar as a disinfectant in small flocks and shows that it is an easy and
inexpensive way to do so. Additionally, farmers harbor less inhibitions or qualms with putting
vinegar or lemon juice in animal water than using bleach, or using chemical disinfectants for
cleaning. This data has shown that organic acids may be used as disinfectants which would allow
individuals who are raising organic poultry for market or personal use to improve and maintain
bird health.
Personal experience in rural areas of Guatemala and Mozambique emphasized the lack of
resources and the need for effective disinfectants. One local described a textbook case of
Pullorum Disease when discussing problems with her flock. Any animal medications or
treatments for disease were unavailable to most of the populace and farm disinfectants were hard
46
to find. However, bleach was available and used by many people in their households for various
purposes. Furthermore, citrus fruits such as lemons and limes grow naturally in many of the
areas visited; it would be possible for the juice to be harvested and used as a potential natural
disinfectant.
Undeveloped areas that do not have access to our commercial disinfectants can use one of
the household alternatives tested in order to decrease Salmonella contamination. As
demonstrated, efficacy is dependent on time and serotype but any decrease in bacterial count is
preferable and these alternatives are often available to even the most rural areas. The data
collected in this experiment should be considered general guidelines, rather than hard and fast
rules.
CONCLUSIONS AND APPLICATIONS
1. Serotypes may respond to disinfectants differently.
2. Salmonella are constantly evolving, so disinfectants may become less effective over time.
3. Disinfectants should be periodically tested for efficacy against intended bacteria.
4. Bacterial load has an impact on disinfectant efficacy.
5. Lower concentrations of disinfectant may or may not be as efficacious as the solution
suggested on disinfectant labels.
6. Longer contact time increases efficacy and in some cases can make up for a lower
strength dilution.
7. When commercial disinfectants are unavailable, alternatives that are often easily found
around the household may be substituted. However, efficacy varies.
47
REFERENCES AND NOTES
1. Saif YM, editor. Diseases of Poultry. 12th ed. Ames: Blackwell Publishing; 2011.
2. Salmonella Homepage | CDC. 2016 [accessed 2016 May 27].
http://www.cdc.gov/salmonella/index.html
3. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance.
Clinical microbiology reviews. 1999 [accessed 2015 Oct 23];12(1):147–79.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=88911&tool=pmcentrez&rendertype
=abstract
4. Kennedy J, Bek J, Griffin D. Selection and Use of Disinfectants. 2000.
5. Jeffrey DJ. Chemicals used as disinfectants . Active ingredients and enhancing additives. Rev
Sci Tech. 1995;14(1):57–74.
6. Centers for Disease Control and Prevention. Eight Multistate Outbreaks of Human Salmonella
Infections Linked to Live Poultry in Backyard Flocks. 2016.
http://www.cdc.gov/salmonella/live-poultry-05-16/
7. Aslam A. Efficacy of selected disinfectants against Salmonella isolates from broiler breeders.
University of Arkansas; 2013. 132 p.
48
Table 1. Disinfectants used and their active ingredients.
Disinfectant Active Ingredients
Virkon-S Potassium peroxymonosulfate…….21.41%
Sodium chloride….1.50%
Virocid
Alkyl dimethyl benzyl ammonium chloride…17.060%
Didecyl dimethyl ammonium chloride…7.8%
Glutaraldehyde…10.725%
Pheno-Tek
Para tertiary amylphenol…8.24%
Ortho benzyl para chlorophenol…5.3%
Ortho phenylphenol…4.96%
Bleach Sodium hypochlorite…8.5%
Lemon Juice Citric acid…3.41%
Vinegar Acetic acid…5%
49
Table 2. Plate count values for S. Enteritidis at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* 26 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T 1 0 0
.5X‡ 0 0 0 0
.25X* T T 0 0
.1X‡ T 245 1 0
.1X* T T T 7
.01X‡ T T T T
VINEGAR X* 0 0 0 0
VIROCID X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* T 260 1 0
.1X‡ T T T 0
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T T
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† T 0 0 0
.001X† T 13 5 0
.0002X† T T T T
* Starting isolate concentration was 9.35 log CFU ‡ Starting isolate concentration was 9.2 log CFU
† Starting isolate concentration was 9.13 log CFU T: too numerous to count
49
50
Table 3. Plate count values for S. Heidelberg at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* T 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T T 4 0
.5X‡ 0 0 0 0
.25X* T T T 37
.1X‡ T 110 0 0
.1X* T T T T
.01X‡ T T T T
VINEGAR X* T 3 0 0
VIROCID X‡ 0 0 0 0
.5X* T T 1 0
.5X‡ 0 0 0 0
.25X* T T T 37
.1X‡ T 24 0 0
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T T
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† 0 0 0 0
.001X† 0 T 31 0
.0002X† T T T T
* Starting isolate concentration was 9.19 log CFU ‡ Starting isolate concentration was 9.6 log CFU
† Starting isolate concentration was 9.2 log CFU T: too numerous to count
50
51
Table 4. Plate count values for S. Kentucky at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* T 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T T 0 0
.5X‡ 0 0 0 0
.25X* T T 12 0
.1X‡ T T 104 0
.1X* T T T T
.01X‡ T T T T
VINEGAR X* 0 0 0 0
VIROCID X‡ 0 0 0 0
.5X* 29 0 0 0
.5X‡ 0 0 0 0
.25X* T T 1 0
.1X‡ T 0 5 0
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T 270
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† 0 0 0 0
.001X† 0 0 0 0
.0002X† T 46 0 0
* Starting isolate concentration was 9.4 log CFU ‡ Starting isolate concentration was 9.4 log CFU
† Starting isolate concentration was 9.1 log CFU T: too numerous to count
51
52
Table 5. Plate count values for S. Typhimurium at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* T 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T 19 0 0
.5X‡ 0 0 0 0
.25X* T T 0 0
.1X‡ T T 2 0
.1X* T T T 3
.01X‡ T T T T
VINEGAR X* 0 0 0 0
VIROCID X‡ 0 0 0 0
.5X* 13 0 0 0
.5X‡ 0 0 0 0
.25X* T 3 0 0
.1X‡ T 1 0 0
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T T
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† 0 0 0 0
.001X† 4 0 0 0
.0002X† T T T 0
* Starting isolate concentration was 10.92 log CFU ‡ Starting isolate concentration was 9.83 log CFU
† Starting isolate concentration was 9.31 log CFU T: too numerous to count
52
53
Table 6. Plate count values for S. Senftenberg at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* 0 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T 1 0 0
.5X‡ 0 0 0 0
.25X* T T 0 0
.1X‡ T 207 0 0
.1X* T T T 27
.01X‡ T T T T
VINEGAR X* 0 0 0 0
VIROCID X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* T 28 0 0
.1X‡ T 5 0 0
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T T
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† 0 0 0 0
.001X† 9 0 0 0
.0002X† T T T T
* Starting isolate concentration was 9.67 log CFU ‡ Starting isolate concentration was 9.15 log CFU
† Starting isolate concentration was 9.33 log CFU T: too numerous to count
53
54
Table 7. Plate count values for S. Montevideo at different time intervals and concentrations of disinfectant
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X* T 0 0 0
VIRKON-S X‡ 0 0 0 0
.5X* T 62 0 0
.5X‡ 0 0 0 0
.25X* T T 2 0
.1X‡ T T 27 1
.1X* T T T 31
.01X‡ T T T T
VINEGAR X* 0 0 0 0
VIROCID X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* T T 0 0
.1X‡ T T T 27
.1X* T T T T
.01X‡ T T T T
BLEACH X* 0 0 0 0
PHENO-TEK X‡ 0 0 0 0
.5X* 0 0 0 0
.5X‡ 0 0 0 0
.25X* 0 0 0 0
.1X‡ T T T T
.1X* 0 0 0 0
.01X‡ T T T T
.01X† 0 0 0 0
.002X† 5 1 0 0
.001X† 133 T 0 0
.0002X† T T T 0
* Starting isolate concentration was 9.19 log CFU ‡ Starting isolate concentration was 11.13 log CFU
† Starting isolate concentration was 9.16 log CFU T: too numerous to count
54
55
Table 8. Number of Salmonella strains each disinfectant reduced and/or killed at each time
MINUTES
MINUTES
5 15 30 60
5 15 30 60
LEMON X 2 6 6 6
VIRKON-S X 6 6 6 6
.5X 0 4 6 6
.5X 6 6 6 6
.25X 0 0 5 6
.1X 0 3 6 6
.1X 0 0 0 4
.01X 0 0 0 0
VINEGAR X 5 6 6 6
VIROCID X 6 6 6 6
.5X 5 5 6 6
.5X 6 4 6 6
.25X 0 3 5 6
.1X 0 4 4 6
.1X 0 0 0 0
.01X 0 0 0 0
BLEACH X 6 6 6 6
PHENO-TEK X 6 6 6 6
.5X 6 6 6 6
.5X 6 6 6 6
.25X 6 6 6 6
.1X 0 0 0 1
.1X 6 6 6 6
.01X 0 0 0 0
.01X 6 6 6 6
.002X 5 6 6 6
.001X 5 4 6 6
.0002X 0 1 1 3
55
56
CHAPTER 3
A survey of the prevalence of internal parasites in chickens shown at Arkansas Fairs
57
A survey of the prevalence of internal parasites in chickens shown at Arkansas Fairs
K. Moyle* F.D. Clark*1, and J.R. Moyle†
*University of Arkansas, Division of Agriculture, Center of Excellence for Poultry Science,
Fayetteville, AR
†University of Maryland Extension, College Park, MD
Primary Audience: Small and Medium Scale Poultry Producers, Hobby Flock Owners,
Veterinarians, Extension Agents
SUMMARY
Backyard and exhibition poultry have been gaining in popularity and as such there has
been a large increase in the number of small flocks. Typically most backyard flocks are allowed
to roam freely for part or all of the day. As these free-range birds interact with the environment
there is an increased risk of exposure to parasitic infection due to factors such as exposure to
wild birds, intermediate hosts such as earthworms, and lack of knowledge. Quite often the
owners are new to poultry and don’t recognize the potential for parasite infection, nor how to
identify and treat it if infection does occur. Four of the most common internal parasites found in
backyard poultry are Eimeria, Capillaria, Heterakis, and Ascaridia. Some research has been
done on the prevalence of parasites in small flocks internationally but very little in the United
States. Coccidia were found in more than half of flocks for two years, and roughly a quarter of
flocks are infected with ascarids. At least a quarter of flocks showed evidence of Capillaria, and
almost a third of flocks were infected with 2 or more species of parasites. This study indicates
that there is a high prevalence of parasite infection in backyard flocks, especially with coccidia.
1 Corresponding author: [email protected]
58
Key words: poultry, hobby flock, parasites, ascarids, coccidia
DESCRIPTION OF PROBLEM
Research and surveys have been done on the prevalence of internal parasites in flocks
internationally [1-2], especially in Africa [3–6], but very little has been done in the United States
[7]. The purpose of this study was to determine the prevalence of common internal parasites in
small poultry flocks in Arkansas.
MATERIALS AND METHODS
Samples were randomly collected every year from the Arkansas-Oklahoma State Fair
held in Ft. Smith, Arkansas, and the Arkansas State Fair in Little Rock for the years 2013-2015.
In 2015 samples were also collected from the Washington County Fair in Northwest Arkansas.
Fresh fecal samples were individually collected from cages then transported to the University Of
Arkansas Center Of Excellence for Poultry Science and analyzed for parasites by performing
fecal flotation per Soulsby [8]. Fecal flotation was performed by mixing 1 gram of fecal material
with 10 ml of saturated sodium chloride solution. A glass coverslip was placed on top of the test
tube and left for at least 20 minutes. The coverslip was placed on a slide and examined under
microscope for presence or absence of parasites.
RESULTS AND DISCUSSION
Over the course of three years, a total of 909 samples from different birds were analyzed,
collected from 279 entrants; each entrant is considered an individual flock. In an effort to
understand the dynamics of parasites in small flocks we analyzed the data for the years
individually in order to track variation in infection. The study found that in backyard flocks
59
shown in Arkansas, the percent of all flocks infected with Ascaridia ranged between 24-28%
over the course of 3 years. Infection with Eimeria ranged between 44-71%; infection with
Capillaria was between 25-34%, and incidence of Heterakis varied between 2-10%. Flocks that
had evidence of more than one parasite present started at 34% but then dropped down to 27% by
2015. (Graph 1)
When the data for the Little Rock State Fair was analyzed, a drop (from 55% to 38% then
back to 66%) was observed in the number of flocks infected with coccidia during the year 2014.
Flock infection with Ascaridia ranged between 24% and 29%, whereas presence of Capillaria
ranged between 27% and 38%. Infection with Heterakis varied between 3-11%. (Graph 2)
Data for the AR/OK State Fair showed an increase in the amount of coccidial infection,
rising from 61% in 2013 to 85% in 2015. No evidence of Heterakis was found in 2014 or 2015.
In 2013 9% of flocks had evidence of Heterakis. Infection with Capillaria jumped from 20% in
2014 to 54% in 2015. (Graph 3)
There are numerous variables that could have influenced the rate of infection for any of
the species surveyed, including sampling error, Extension hobby flock outreach programs,
management and husbandry, bird housing, bird health, etc. Another major variable is weather,
which influences the quality of the bedding, be it litter or the ground outside. 2014 was a record
cool year with drier than normal conditions during the latter half of the year [9], when samples
were collected. In contrast, the year 2013 was average for temperatures and rainfall [10] while
2015 was warmer and wetter than the 2 years previous [11]. A study done by Permin et al. [3]
examined rural scavenging poultry in Tanzania to see if there was a difference in prevalence of
parasites during the dry season versus the wet season. Ascaridia galli, Heterakis species, and
60
Capillaria species were all identified in the flocks. Although all birds examined were infected by
at least one species of parasite, the study did not find a correlation between season and mean
worm burdens, or season and prevalence. In contrast, a study of organic free range layer systems
in Germany [2] found that risk of infection with H. gallinarum, A. galli, and Capillaria was 50%
higher in the summer. A study of scavenging poultry in two rural areas with different climates in
Zimbabwe [4] found a lower prevalence of Ascaridia in the warm humid area than in the dry, hot
area. However, the study also found a slightly higher prevalence of Capillaria in the warm
humid area than in the dry, hot area. The prevalence of Heterakis was similar for both locations.
Although weather does seem to have an effect on parasite prevalence, the influence it has seems
to fluctuate, most likely depending on various other factors.
Although it is commonly thought that round worms are a bigger issue than thread worms
(F. D. Clark, personal communication, 2016), this data indicates that thread worms are slightly
more prevalent in small backyard flocks in the state of Arkansas.
The low incidence of Heterakis found in this study is similar to data found by Wilson et
al. [7] in their study of commercial broiler chickens from 2 companies. In that study, 7.15% of
farms from company A were infected with H. gallinarum and 1.9% of farms from company B
were infected. The study also found 37.3% of farms from company A to be infected with A.
galli, whereas farms from company B only showed 3.9% of farms being infected. These surveys
of poultry in Arkansas show that A. galli is a much bigger problem than H. gallinarum.
This study found there is a high prevalence of parasite infection in backyard and urban
poultry flocks. The data shows coccidia are especially widespread in Arkansas.
61
CONCLUSIONS AND APPLICATIONS
1. Ascaridia, Eimeria, and Capillaria are common in backyard flocks in Arkansas.
2. Evidence of multiple parasites present in a flock is also relatively common.
3. Extension programs to educate small flock owners about internal parasites and how to
treat and prevent them could prove beneficial.
62
REFERENCES AND NOTES
1. Bhat SA, Khajuria JK, Katoch R, Wani MY, Dhama K. Prevalence of Endoparasites in
Backyard Poultry in North Indian Region: A Performance Based Assessment Study. Asian
Journal of Animal and Veterinary Advances. 2014 [accessed 2016 Jun 2];9(8):479–488.
http://scialert.net/fulltext/?doi=ajava.2014.479.488
2. Kaufmann F, Daş G, Sohnrey B, Gauly M. Helminth infections in laying hens kept in organic
free range systems in Germany. Livestock Science. 2011 [accessed 2016 May 19];141(2-3):182–
187. http://www.sciencedirect.com/science/article/pii/S1871141311002034
3. Permin A, Magwisha H, Kassuku AA, Nansen P, Bisgaard M, Frandsen F, Gibbons L. A
cross-sectional study of helminths in rural scavenging poultry in Tanzania in relation to season
and climate. J HELMINTHOL. 1997;71(3):233–240.
4. Dube S, Zindi P, Mbanga J, Dube C. A Study of Scavenging Poultry Gastrointestinal and
Ecto-parasites in Rural Areas of Matebeleland Province, Zimbabwe. 2010 [accessed 2016 Jun 1].
http://ir.nust.ac.zw/xmlui/handle/123456789/85
5. Offiong EEA, Obioku OE, Umoh JU, Essien CA, Idiong NB. A Survey of Gastrointestinal
Helminthes of Local Chickens in Abak Local Government Area of Akwa Ibom State.
International Journal of Sciences: Basic and Applied Research (IJSBAR). 2013 [accessed 2016
Jun 2];9(1):1–4.
http://gssrr.org/index.php?journal=JournalOfBasicAndApplied&page=article&op=view&path%
5B%5D=952
6. Nnadi PA, George SO. A Cross-Sectional Survey on Parasites of Chickens in Selected
Villages in the Subhumid Zones of South-Eastern Nigeria. Journal of Parasitology Research.
2010;2010.
7. Wilson KI, Yazwinski TA, Tucker CA, Johnson ZB. A Survey into the Prevalence of Poultry
Helminths in Northwest Arkansas Commercial Broiler Chickens. Avian Diseases.
1994;38(1):158–160 .
8. Soulsby EJ. Helminths, Arthropods and Protozoa of Domesticated Animals. 7th ed.
Philadelphia: Lea & Febiger; 1982.
9. US Department of Commerce NNWS. NWS Little Rock, AR - Arkansas Yearly Climate
Summary (2014)/Pg1. [accessed 2016 Jun 1]. http://www.srh.noaa.gov/lzk/?n=2014.htm
10. US Department of Commerce NNWS. NWS Little Rock, AR - Arkansas Yearly Climate
Summary (2013)/Pg1. [accessed 2016 Jun 1]. http://www.srh.noaa.gov/lzk/?n=2013.htm
11. US Department of Commerce NNWS. NWS Little Rock, AR - Arkansas Yearly Climate
Summary (2015)/Pg1. [accessed 2016 Jun 1]. http://www.srh.noaa.gov/lzk/?n=2015.htm
63
Graph 1. Percent of flocks infected with parasites from data collected for the years 2013-2015 at
the AR-OK Fair, Arkansas State Fair, and Washington County Fair.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Ascaridia Eimeria Capillaria Heterakis Multiple
Combined Percent of Flocks Infected
2013
2014
2015
64
Graph 2. Percent of flocks infected with parasites from data collected at the Arkansas State Fair
in Little Rock during the years 2013-2015.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Ascaridia Eimeria Capillaria Heterakis Multiple
Arkansas State Fair
2013
2014
2015
65
Graph 3. Percent of flocks infected with parasites from data collected at the Arkansas Oklahoma
State Fair during the years 2013-2015.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ascaridia Eimeria Capillaria Heterakis Multiple
Arkansas Oklahoma State Fair
2013
2014
2015
66
Graph 4. Percent of flocks infected with parasites from data collected at the Washington County
Fair in 2015.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ascaridia Eimeria Capillaria Heterakis Multiple
Washington County Fair
67
CHAPTER 4
Conclusion
68
CONCLUSION
Salmonella and internal parasites have a major impact on people and poultry worldwide.
Knowledge as to species or serotype, prevalence, prevention, and removal are important in order
to maintain health and productivity.
The first section of this thesis explored the efficacy of 3 commercial disinfectants and 3
common household compounds against various serotypes of Salmonella. The research showed
that knowledge of serotype present in a location or flock is important to determine which
disinfectant would be best, as serotypes respond differently. To make choosing a disinfectant
more difficult, however, Salmonella are evolving over time and resistance to chemicals that were
previously effective is possible. Even the best disinfectant may be ineffective when the bacterial
load is too high or if organic matter is present, so cleaning before disinfection is advised. The
data showed that lower dilutions of disinfectant solutions can maintain efficacy if contact time is
increased; however, there are limitations for how much each disinfectant can be diluted before it
becomes completely ineffective. For farmers in rural or international locations that are unable to
access commercial disinfectants, or for those people who want to avoid chemicals that they view
as unnatural, bleach, lemon juice, and vinegar are possible alternatives.
Bleach is highly effective even at low concentrations, works rapidly, is inexpensive, and
readily available. Because of these factors, it can be reasonably presumed that the average farmer
would not scientifically measure out amounts of bleach; they would most likely measure by
using the cap from the bottle, or by the ‘glug’ method. A capful of bleach from the bottle used in
this experiment contained 12 ml of fluid, and although the size of a glug is variable, it is safe to
presume that most would contain at least 24 ml of liquid, equivalent to 2 capfuls. The data
demonstrates that bleach will kill all the bacteria tested after 15 minutes at a solution of one part
69
bleach, 500 parts water (0.002X). Therefore, one capful of bleach in 1.5 gallons of water would
be enough to completely kill after 15 minutes, and it would kill most of the bacteria present after
only 5 minutes. One capful of bleach and 3.2 gallons of water (0.001X) would reduce bacteria
after 5 minutes, and completely kill after 30. However, one cap of bleach for 3 gallons often does
not seem like much, so most farmers would use the glug method. Just 1 glug for 3 gallons would
give the 0.002X concentration, killing after only 15 minutes. More glugs would continue to
increase the concentration, improving efficacy and reducing kill time.
Lemon juice and vinegar are not as effective as commercial disinfectants or bleach and
are highly influenced by the serotype, but are still capable of reducing bacterial counts under
certain conditions. For those that do not have better resources, lemon juice and vinegar are
possible alternatives. They should be used at higher concentrations. Further research can look
into the efficacy of combining these two acids to determine if there is a synergistic effect.
There are various studies that can be performed following this data, looking further into
possible alternatives that could be used in developing areas. Examples of possible items to try
include various sodas, such as Sprite and Coca-Cola, and slaked lime. An additional real world
application to examine would be the antibacterial effect of leaving items out in the sun for
various periods of time.
The second portion of this thesis discusses a survey of backyard flocks in Arkansas and
the prevalence of four common internal parasites. Data revealed that over half of flocks were
positive for coccidia. Over the course of 3 years, a quarter of flocks showed evidence of
Ascaridia and Capillaria. Prevalence of Heterakis remained below 10%. Between a quarter and
a third of flocks have concurrent infection with multiple parasites. This study revealed that
70
internal parasites in Arkansas are more common than previously thought, and that coccidia are
especially prevalent.
Future research can look at the impact that length of time has on bird health. Some
farmers have been exhibiting birds at fairs for years, whereas some are kids just beginning their
journey. A survey conducted in conjunction with the internal parasite data could give more
information as to how knowledgeable farmers are about the health of their birds and what they
have or have not done in order to maintain their flocks.
While these studies did not examine prevalence of Salmonella in flocks, the increased
incidence of health problems in small flocks, including internal parasites, does necessitate further
study. A preliminary study looking at Mycoplasma synoviae, M. gallisepticum, and Salmonella
incidence in backyard flocks has been started by Extension but the data is not yet complete. Thus
far the incidence of Salmonella has been very low; however, the birds sampled have all been
adults while the majority of outbreaks have been linked to very young chicks. Additional studies
may be of benefit after the conclusion of the preliminary survey.
This data should allow anyone who keeps poultry or works in the poultry industry to have
a better understanding of how to maintain the health of their birds. Additional knowledge of
disinfectant efficacy allows farmers and others to choose a compound that will fit their situation.
In decreasing the prevalence of Salmonella on their farm, they should be able to improve not
only the health of their flocks but the health of themselves and their families, due to the zoonotic
nature of the bacteria. Familiarity with the frequency of internal parasite infection should allow
farmers to be proactive, such as decreasing the conditions required for contact and spread.
Additionally, farmers will be able to medicate their birds for a specific condition rather than
71
using a broad spectrum treatment for diseases the birds do not have. By becoming aware of the
common diseases in an area, being proactive, and knowing how to maintain clean premises,
farmers will be better able to maintain their health and the health of their flocks.